Affiliation

Abstract

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell-cell contacts. Cell migration activity is increased by 8-17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.

Formation of membrane extensions and microspikes by fascin transfection. Parental LLC-PK1 and fascin-transfected…

Figure 2

Formation of membrane extensions and microspikes by fascin transfection. Parental LLC-PK1 and fascin-transfected cells were stained with FITC-phalloidin to examine F-actin organization at the periphery. (A) Image of parental LLC-PK1 cells; (B) image of fascin-transfected cells taken at a lower focal plane; (C) image of fascin-transfected cells taken at a higher focal plane. Note that fascin transfection induces the membrane extension at lower surfaces (arrowhead in B), as well as the formation of long and thick microspikes on upper surfaces (asterisk in C). Circumferential actin bands of parental LLC-PK1 cells appear to transform into ring-like structure (arrowhead in C) from which microfilament bundles are radiating. Bar, 10 μm.

Figure 3

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Disorganization of cell–cell contacts shown…

Figure 3

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Disorganization of cell–cell contacts shown by fascin transfectants. Cells were double-labeled with fluorescent…

Figure 3

Disorganization of cell–cell contacts shown by fascin transfectants. Cells were double-labeled with fluorescent phalloidin and anti-β-catenin antibody. (A and B) LLC-PK1 cells; (C and D) fascin-transfected cells. (A and C) F-actin localization by phalloidin staining; (B and D) β-catenin localization. Note that cell–cell contacts are disorganized in fascin transfectants. The “rings” of transfectants (arrowhead in C and D) are stained with both the β-catenin antibody and phalloidin, confirming that they are derived from circumferential actin bands as a result of disorganization of cell–cell contacts of parental LLC-PK1 cells. Bar, 10 μm.

Organization of fascin and β-catenin in parental LLC-PK1 cells. (A and C) Fascin…

Figure 5

Organization of fascin and β-catenin in parental LLC-PK1 cells. (A and C) Fascin localization; (B and D) β-catenin localization. (C) and (D) are high magnification images corresponding to (A) and (B), respectively. Note that fascin is localized in short microspikes radiating from the adhesion belts while β-catenin is continuously localized on the adhesion belts (arrowhead). Bar, 10 μm.

Figure 6

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Localization of fascin and β-catenin…

Figure 6

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Localization of fascin and β-catenin in fascin-transfected cells. Intact (A and B) or…

Figure 6

Localization of fascin and β-catenin in fascin-transfected cells. Intact (A and B) or Triton-permealized (C and D) fascin transfectants were double labeled with antifascin and anti-β-catenin antibodies. (A and C) fascin; (B and D) β-catenin. In intact cells (A and B), fascin staining is not entirely diffuse but appears particulate (A). In addition, fascin shows the localization in membrane extensions (arrowhead in A). Triton permealization makes fascin’s localization in microvilli and membrane protrusions very clear (C). Fascin localizes in long and thick microvilli (arrowhead in C) radiating from “rings,” as well as in membrane extensions (arrow in C). Note that less β-catenin is found where more fascin microfilament bundles are developed (arrowhead with asterisk in C and D). Bar, 10 μm.

Decrease in the β-catenin expression in fascin-transfected cells. Total cell lysates of parental…

Figure 8

Decrease in the β-catenin expression in fascin-transfected cells. Total cell lysates of parental LLC-PK1 (lane 1), mock-transfected LLC-PK1 (lane 2), and two clones (lane 3, clone 1; lane 4, clone 2) of fascin transfectants were immunoblotted with antibodies against fascin, β-catenin, or β-tubulin. Fascin antibody (55K-2) detected both endogenous pig fascin, as well as exogenous human fascin as indicated. Clones 1 and 2 express human fascin 12 times and 17 times more than the level of endogenous fascin. In contrast, β-catenin expression of clones 1 and 2 is decreased to 33% and 20%, respectively. Mock-transfected cells show no decrease in β-catenin expression. β-tubulin level was used as an internal control. H, Human fascin; P, pig fascin; β-Cat., β-catenin; β-Tub, β-tubulin.

Figure 9

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Microinjection of fascin into LLC-PK1…

Figure 9

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Microinjection of fascin into LLC-PK1 cells causes morphological alterations similar to those found…

Figure 9

Microinjection of fascin into LLC-PK1 cells causes morphological alterations similar to those found in DNA transfection. Asterisks indicate microinjected cells. (A–C) Induction of microvilli on apical surfaces. Two LLC-PK1 cells were microinjected with human fascin, fixed with formaldehyde, and stained with rhodamin phalloidin (B) and the 55k-2 fascin antibody (A). (C) Phase contrast. The microinjected cells develop more microvilli on their apical surfaces (B). Note that longer microspikes are found radiating from circumferential bands (arrowheads). Fascin staining in (A) is not of good quality; it is only for the identification of injected cells because the fascin antibody (55k-2) does not work well with formaldehyde-fixed cells and gives a weak staining with nonspecific background. (D–F) Induction of membrane protrusions at basolateral surfaces by fascin microinjection. Four LLC-PK1 cells were microinjected, fixed with methanol, stained with the fascin antibody, and photographed at two different focal planes. (D) Basolateral surface; (E) apical surface; (F) phase contrast. Note that microinjected cells extend basolateral membranes (indicated by arrowheads in D). (G–I) Increase in phalloidin staining by microinjection with fascin. A cell was injected with human fascin together with FITC-dextran, stained with rhodamin phalloidin, and photographed using an AT200-cooled CCD camera (Photometrics). Deconvoluted images of panels H and I were obtained using MicroTome software. (G) FITC-dextran; (H) F-actin organization at a higher focal plane; (I) F-actin at a lower focal plane. F-actin staining is higher with injected cells (asterisk, H and I). Note that circumferential actin bands are partially disorganized (H, indicated by arrowhead) and that microvilli are more developed in the injected cell (H). Bar, 10 μm.

Figure 10

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Induction of membrane protrusions by…

Figure 10

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Induction of membrane protrusions by microinjection of fascin into REF-52 cells. (A) Recombinant…

Figure 10

Induction of membrane protrusions by microinjection of fascin into REF-52 cells. (A) Recombinant human fascin was microinjected into REF-52 fibroblastic cells and stained with fascin antibody. (B) As a control, caldesmon was injected and stained with caldesmon antibody. Note that fascin microinjection causes numerous protrusions along all sides of the cell periphery of REF-52 cells (arrowhead in A). Bar, 10 μm.